Dengue viruses are divided into four antigenically related serotypes, called dengue virus serotypes 1, 2, 3, and 4. Complete or partial nucleotide sequences of the dengue 1, 2, 3, and 4 type virus have been published (Chamber, T. J., Hahn, C. S., Galler, R. And Rice, C. M. 1990. Annu. Rev. Microbiol., 44, 649, Zhao et al., 1986, Virology 155, 77-88).
Dengue virus, with its four serotypes Den-1 to Den-4, is the most important member of the Flavivirus genus with respect to infections of humans producing diseases that range from flu-like symptoms to severe or fatal illness, dengue haemorrhagic fever with shock syndrome. Dengue outbreaks continue to be a major public health problem in densely populated areas of the tropical and subtropical regions, where mosquito vectors are abundant. Therefore, there is a substantial need for the development of prophylactic vaccines. Previous efforts to prepare live candidate dengue vaccines were mainly based on classical attenuation of dengue virus by serial passage in animals or in cultured cells of non-natural hosts. However, this approach has not been consistently successful in producing attenuated vaccine strains. Available data indicate that recovery and protective immunity after dengue virus infection are correlated to the development of high titres of virus neutralising antibodies. However, this immunity is homotypic mediating resistance to the same virus serotype only. Moreover, individuals immune to one dengue virus serotype may be even at higher risk of developing severe dengue illness if reinfected with another serotype. To overcome these problems an ideal vaccine should therefore induce solid immunity against all four dengue virus serotypes.
The flavivirus genome consists of a single-stranded positive sense RNA molecule. The single encoded open reading frame (ORF) is translated into a polypeptide which is cleaved co- and post-translationally into at least 11 proteins. The order of proteins encoded in the ORF is 5′-C-preM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′(Venugopal, K., and Gould, E. A., 1994, Vaccine, Vol. 12, No. 11).
Previous results from vaccine trials in animal models indicated that immune responses to structural preM and E proteins, or non-structural NS1 proteins were fully protective against a lethal challenge with homotypic dengue virus (Zhao et al. 1987 J. Virol. 61:4019; Bray et al. 1989 J. Virol. 63:2853; Falgout et al. 1990 J. Virol. 64:4356; Fonseca et al. 1994 Vaccine 12:279; Srivastava et al. 1995 Vaccine 13:1251).
The preM protein (18-19 kDa) is a precursor of the structural protein M, which is formed by cleavage and removal of the N-terminal (pre) segment, by a process presumed to be linked to maturation of the envelope glycoprotein and the development of virus infectivity.
The E glycoprotein (53-54 kDa) is an outer structural protein of the dengue virus. It exhibits a number of biological activities including receptor binding and membrane fusion, and is the target for neutralising antibodies and T-helper cells. The E protein is a typical membrane glycoprotein with a C-terminal that spans the membrane.
The non-structural protein NS1 (39-41 kDa) is also modified by glycosylation. It is derived from the ORF by N-terminal signalase cleavage and C-terminal cleavage involving a protease. NS1 may be involved in the assembly and release of virions. It is found on the cell surface and in the culture medium of infected cells. During the course of infection, NS1 protein evokes a strong antibody response which protects the host against challenge with flaviviruses, presumably through a complement mediated pathway, although recently is has been suggested that other mechanisms such as antibody-dependent cell cytotoxicity may also be responsible (Venugopal, K., & Gould, E. A., 1994, Vaccine, Vol. 12, No. 11).
Knowledge about the molecular biology of flaviviruses rapidly increased during the last decade, and led to the application of recombinant techniques for the production of new vaccine candidates. These approaches have included E. coli fusion proteins, baculo virus produced recombinant proteins, and live recombinant vaccinia viruses. Notably, the vaccinia approaches have given promising results. In mice, total protection against lethal challenge with dengue virus has been achieved after immunisation with recombinant vaccinia viruses expressing structural and/or non-structural genes of dengue virus (J Gen Virol, 1988, 69: 2102-7).
There is still the need for the development of a safe and an effective vaccine with a major goal in the prevention, and perhaps the treatment, of DF and DHF/DSS in humans. As mentioned above only approaches using recombinant vaccinia virus have given so far promising results. However, occurrence of rare adverse reactions to smallpox vaccination and the increased susceptibility of immunodeficient individuals has made further attenuation and improved safety a priority for human vaccines based on recombinant vaccinia virus.
Modified vaccinia virus Ankara (MVA), is a host range restricted and highly attenuated vaccinia virus strain, which is unable to multiply in human and most other mammalian cell lines tested. But since viral gene expression is unimpaired in non-permissive cells recombinant MVA viruses may be used as exceptionally safe and efficient expression vectors.
The modified vaccinia virus Ankara (MVA) has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H. (1975) Infection 3, 6-14; Swiss Patent No. 568, 392). The MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures de Microorganisms, 25, rue du Docteur Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. I-721. The MVA virus has been analyzed to determine alterations in the genome relative to the wild type CVA strain. Six major deletions of genomic DNA compared with the wild type CVA (deletion I, II, III, IV, V, and VI) totalling 31,000 base pairs have been identified (Meyer, H., Sutter, G. and Mayr A. (1991) J. Gen. Virol. 72, 1031-1038). MVA is further distinguished by its great attenuation, that is to say by diminished virulence or infectivity while maintaining good immunogenicity. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167,375-390 (1987), Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 (1974)). During these studies in over 120,000 humans, including high risk patients, no side effects were associated with the use of MVA vaccine. MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virions. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B. (1992) Proc. Natl. Acad. Sci. USA 89,10847-10851). Recently, novel vaccinia vector systems were established on the basis of MVA, having foreign DNA sequences inserted at the site of deletion III within the MVA genome (Sutter, G. and Moss, B. (1995) Dev. Biol. Stand. Basel, Karger 84,195-200). Another approach used, except less successfully (Scheiflinger et al., 1996, Arch Virol 141, 663-669) the tk gene within the MVA genome (U.S. Pat. No. 5,185,146).
According to the present invention, DNA sequences (genes) which code for dengue antigens are introduced, with the aid of DNA recombination techniques, into the genome of MVA. When the DNA sequence encoding the dengue antigen is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, e.g. one of the above mentioned deletions, the newly produced recombinant MVA will be infectious, that is to say able to infect foreign cells and it will express the integrated DNA sequence. The recombinant MVA according to the invention will be useful as extremely safe live vaccines for the treatment or prophylactics of infectious diseases.